The chemical energy for sperm motility is provided by the hydrolysis of ATP within the flagellum. To elucidate the mechanism of sperm motility it is necessary to isolate, characterize, and localize the movement-related ATPase(s) of the flagellum. To extract the ATPase(s) from mammalian sperm, uncontaminated by ATPases from membranes and disrupted mitochondria, procedures were developed for the preparation of plasmalemmae-free, mitochondria-free flagella isolated from bull sperm and from human sperm. Alterations in sperm morphology and the degree of mitochondrial removal were monitored throughout the procedure by light microscopy and by transmission electron microscopy (TEM) of negatively stained samples. TEM of thin sectioned samples was used to evaluate the effect of each treatment on internal flagellar ultrastructure and confirmed that after the mitochondria are gone the outer dense fiber/axonemal complex, and especially the dynein arms, are maintained. Although the total ATPase activity of isolated bull sperm flagella decreases after removal of the mitochondria, a Mg ion-ATPase of high specific activity is extractable from the decapitated, plasmalemmae-free, mitochondria-free flagella. This indicates that the movement-related ATPase(s) of bull sperm flagella is preserved when the mitochondria are removed by our procedure. BIBLIOGRAPHIC REFERENCES: Young, L.G. and E.B. Smithwick. 1976. Effects of mechanical and chemical disruption on the ATP-phosphohydrolase activity and ultrastructure of sperm flagella. Exp. Cell Res. 102:179-190. Smithwick, E.B. and L.G. Young. 1977. Microscopic evaluation of the preparation of decapitated plasmalemmae-free, mitochondria-free bull sperm flagella. TSEM Newsletter 8(2):29 (Abstract).